Complete or draft versions of genome sequences have been obtained in a variety of organisms, including human (Hattori et al., 2000; Lander et al., 2001; McPherson et al., 2001; Sachidanandam et al., 2001; Venter et al., 2001), and a large fraction of the genes are also mapped to chromosomal regions. A current challenge is to annotate gene maps with phenotypic information that imparts functional meaning to the genomic sequences. Thus, genomic research efforts have shifted to systematic determination of gene function (e.g., via analysis of mutant phenotypes). See e.g., Parinov & Sundaresan, 2000; Beckers & Angelis, 2001; Rossant & McKerlie, 2001; Yaspo, 2001. The ability to link a phenotype with one or more genes responsible for that trait provides opportunities for new diagnostics and treatments of genetic diseases.
Large-scale random mutagenesis approaches have generally relied on creating or inducing genetic modifications, the impact of which are evaluated in the context of a complete organism. Screening approaches for the selection of both dominant and recessive mutations are available in plant and animal model organisms, including Drosophila melanogaster (Gans et al., 1975; Nusslein-Volhard & Wieschaus, 1980), Caenorhabditis elegans (Brenner, 1974; Kemphues et al., 1988), Arabidopsis thaliana (Budziszewski et al., 2001; McElver et al., 2001), Danio rerio (Driever et al., 1996; Haffter et al., 1996), and Mus musculus (see citations below). For recovery of mutations that confer early organismal lethality, methods have been developed for the systematic generation of mosaic animals bearing homozygous mutant clones. See e.g., Xu et al., 1995; Duffy et al., 1998.
For insights into human disease, the mouse is an experimental genetic system of choice because its genes, biochemical pathways, and physiological organ functions are closely related to those in humans. Random mutagenesis screens in mouse initially focused on screens for dominant mutations that result in viable, clinically relevant phenotypes (Hrabe de Angelis et al., 2000; Isaacs et al., 2000; Nolan et al., 2000). Genome-wide screens that select recessive mutations (Kasarskis et al., 1998; Fahrer et al., 2001) and screens based on mosaic analysis (Liu et al., 2002) have more recently been undertaken.
Since phenotypic screens have relied on whole organism analysis, systematic mutagenesis studies have been limited to model genetic organisms. Thus, a functional genomics approach has generally not been available in most organisms, including humans, agriculturally important plants and animals, domestic animals, pathogens, etc. However, genomic sequencing has been accomplished or is currently sought in many non-model organisms, and functional annotation is similarly valuable.
A reverse genetics strategy called double-stranded RNA interference has been developed recently as a method for functional analysis in non-model organisms. According to this approach, double-stranded RNA is used to target specific RNA transcripts for degradation, thereby leading to a loss of gene function. Since the double-stranded RNA is prepared based on known sequence, the link between gene and phenotype is already known.
RNA interference is a silencing phenomenon that is manifest in plants, animals and fungi, and therefore enables systematic functional analysis of any organism for which genomic sequence data is known. See Zamore, 2001; Carthew, 2001. This strategy has been adopted for genome-wide analysis in C. elegans (Bargmann, 2001). Despite its utility in diverse organisms, RNA interference is limited to loss-of-function analysis. Thus, this strategy is inapplicable for the discovery of disease-related mutations resulting from increased or otherwise altered gene function.
Thus, current and long-felt needs in the field include strategies for rapid phenotyping and gene mapping that can be performed in any species. The presently claimed subject matter discloses methods for generating, phenotyping, and mapping mutations in vitro, and thus addresses the current and long-felt need in the art for the same.